1. Field of the Invention
The present invention relates to novel reagents and methods which utilize a universal in vivo delivery system based on the use of polymer: polymer recognition and binding to deliver active compounds to target analytes for diagnostic and therapeutic purposes.
2. Brief Description of the Prior Art
Hormones (Varga, J. M., Asato, N., Lande, S, and Lerner, A. B. (1977) Nature 267: 56-58), lectins (Kitao, T. and Hattori, K. (1977) Nature 265: 81-82), and antibodies are among the reagents that have been employed as delivery agents in a variety of systems developed for the purpose of specifically delivering compounds to in vivo targets for diagnostic or therapeutic purposes. However, monoclonal antibodies are currently preferred. Recognition and delivery of therapeutic or diagnostic reagents using these antibodies has been accomplished through a single-step procedure in which the therapeutic or diagnostic reagent is directly conjugated to the monoclonal antibody. These conjugated monoclonal antibodies do not, however, represent a perfect delivery system. Monoclonal antibodies require a long period of time to reach equilibration with cell surface epitopes in vivo and may require up to several days to achieve optimal binding to a particular tissue (Primus, F. J., Wang, R. H., Goldenberg, D. M., et al. (1973) Cancer Res. 33: 2977-2982; Moshakis, V., McIlhinney, R. A. J., Raghavan, D., et al. (1981) Br. J. Cancer 44: 91-90). Thus, the use of monoclonal antibody delivery systems for radioimaging or therapeutic drug delivery, e.g., radioactively labeled monoclonal antibodies or drug-monoclonal antibody conjugates, may result in damage to untargeted tissues due to their lack of specificity and to the lengthy exposure times required. Clearance of these antibody conjugates may also result in some degree of kidney damage due to the large excess of these reagents that must be employed in order to achieve optimal results. In addition, the long equilibration period required for optimal monoclonal antibody binding precludes the use of short-lived radioisotopes in these delivery systems.
A major focus of current research has been to improve targeting of therapeutic or diagnostic reagents to internal target sites, such as to solid tumors or specific organs. One objective of this targeting is to enhance the effectiveness of these conjugated monoclonal antibody reagents by concentrating them at the target site, thereby minimizing their effects on non-target sites. For example, if a reagent is used for therapeutic purposes, such as to treat a solid tumor, improved targeting provides more effective dosing at the target site with fewer non-tumor related side effects. Similarly, where the reagent is a radionuclide used for radioimaging, improved targeting provides enhanced contrast between the target and background areas due to reduced background levels of the radionuclide. Another objective of this research has been to reduce the time required for delivery of the diagnostic or therapeutic reagent to target sites. This has been accomplished using a multi-step procedure in which antibody binding to target sites and reagent delivery are performed in separate steps. The advantage of such a procedure is that the targeting reagent can be allowed to equilibrate for a lengthy period of time without exposing the patient to a potentially harmful reagent. An equilibration period of one or more days will also result in greater specificity and lower backgrounds since excess targeting reagent is likely to be cleared during this period of time.
An example of a multi-step drug delivery procedure which has been used to deliver drugs to cells in vitro is disclosed in an article by Urdai and Hakomori (Urdai, D. L. and Hakomori S. I (1980) J. Biol. Chem. 255: 10509-10516). In this in vitro system, avidin was used as a bridge to link biotinylated, target-specific antibodies to either biotin-drug or biotin-drug-liposome drug carrier reagents.
Godfrey, et al. (Godfrey, W., Doe, B., Wallace, E. F., Bredt, B., and Wofsy, L. (1981) Experimental Cell Research 135: 137-45) also disclose the use of a biotin-avidin bridge to link antibodies to biotinylated drug-carrying liposomes or red blood cell ghosts in a similar multi-step in vitro assay for identifying specific types of cells.
Trubetskoy, et al. (Trubetskoy, V. S., Berdichevsky, V. R., Efremov, E. E., and Torchilin, V. P. (1987) Biochemical Pharmacology 36: 839-842) also recognized that a multi-step procedure results in a more effective delivery of therapeutic or diagnostic reagents to targets. The procedure they propose involves sequential administration of a targeting reagent, i.e., a biotinylated antibody which specifically recognizes and binds to the target site and which accumulates at the targeted tissue, followed by administration of avidin and a biotinylated liposome drug-carrying system which is capable of coupling specifically to the target-bound biotinylated antibody through the avidin bridge. The “bridge” molecules are defined by Trubetskoy as being bifunctional entities in that they have affinity for both the target binding reagent and the drug-carrying reagent. These “bridge” molecules can be “natural polyvalent macromolecules” which are “capable of binding two or more ligands, or synthetic conjugates obtained from molecules possessing two different types of affinty.” In this paper the conjugate of the antibody, biotin, and avidin serves as the bridge between the target antigen and the biotinylated liposome, according to Trubetskoy. This system differs from one in which highly specific binding of arrays of like polymer units are used to link targeting and drug-carrying reagents.
In an alternate in vivo delivery system which employs biotin-avidin linkages, Goodwin, et al. (Goodwin, D. A., Meares, C. F., McCall, M. J. and McTigue, M., (1987) J. Nucl. Med. 28: 722) disclose a multi-step procedure wherein a conjugate comprised of streptavidin or avidin, a biotinylated EDTA chelate, and a radioactive metal (111In) is used to enhance the localization and delivery of the chelated metal isotope to the liver. This report further demonstrates the feasibility of using a lengthy equilibration period, i.e., 20-24 hours, for equilibration of the targeting entity, avidin, to the target tissue prior to addition of a biotinylated metal chelate complex. This system is further discussed in a subsequent publication by these workers (see, Goodwin, D. A. Meares. C. F., McCall, M. J. McTigue, M., Chaovapong, W., Levy, R. and Starnes, C. (1987) J. Nucl. Med. 28: 561), wherein the authors teach the use of a multi-step in vivo targeting system for mice in which monoclonal antibody-avidin conjugates are pre-equilibrated with target cells in vivo, then subsequently bound to biotinylated radioactive metal chelates prior to radioimaging. This paper also teaches the use of a clearing step with biotinylated human transferrin to remove excess monoclonal antibody-avidin conjugates from the blood. This system is more fully described in Eur. Pat. Pub. No. 0 251 494, published on Jan. 7, 1988, to Goodwin. D. A. Meares, C. and McCall, M. A drawback of this procedure is that high background levels result from the endogenous biotin present in the organism which can result in non-specific binding of the monoclonal antibody-avidin conjugate.
A different multi-step procedure which has been used to image murine tumors was disclosed by Goodwin, et al. (Goodwin, D. A., Meares, C. F., McCall, M. J., McTigue, M. and Chaovapong, W. (1988) J. Nucl. Med. 29: 226-234). In this procedure, antibody to a portion of a metal chelate complex was administered to mice and allowed to passively accumulate in the animal over a period of 20-24 hours. Excess antibody circulating in the blood was then removed by a brief chase with the antigen linked to human transferrin. Radioactively labeled chelate complexes were administered intravenously 1-3 hours before imaging. This procedure represents an improvement over previous radioimaging techniques because it allows for lengthy equilibration periods with a non-radioactive targeting agent, removal of excess targeting reagent, more rapid, specific, and efficient binding of chelated radioactive metals, and shorter periods of exposure to radioactive materials. However, its efficacy is reduced by the loss of antibody-bound chelated radioisotopes, since binding of these antibodies to metal chelates is very weak, resulting in weak signals and high background levels. Moreover, the antibodies are not specifically directed to a single organ, tissue, or cell type, further resulting in non-specific distribution of the radioactive chelate and high background levels.
The use of the above-described delivery systems for therapeutic or diagnostic reagents also presents a health hazard to the patient because high levels of such reagents must be used in these procedures. Thus, there is a need to develop a delivery system which exhibits high specificity for target sites, low background levels, and which also minimizes unnecessary exposure of patients to radioactive ions, or other hazardous materials.
A different type of bridging entity for use in a two-step universal in vitro system for detecting target analytes in a sample is taught in U.S. patent application Ser. No. 08/032,769, filed on Mar. 16, 1993, which is a continuation of U.S. patent application Ser. No. 07/607,787, filed on Oct. 26, 1990, now abandoned, which is a continuation of U.S. patent application Ser. No. 06/922,757, filed on Oct. 24, 1986, now abandoned, which is a Continuation Application of U.S. patent application Ser. No. 491,929, filed on May 5, 1983, now abandoned, both assigned to the instant assignee, which are incorporated by reference herein and made a part thereof. European Patent Application No. 84 10 5028.9-2110 was filed on May 4, 1984, based upon the priority document, U.S. patent application Ser. No. 06/491,929 (filed on May 5, 1983), and published as EP 0 128 332 A1 on Dec. 19, 1984. The European Patent Office issued a Communication under Rule 51(4) EPC on Aug. 20, 1994 indicating its intention to grant a patent thereon. In the first step of the process, a bridging reagent is administered and allowed to achieve binding equilibrium with the target. This reagent has two components. One of the components recognizes the target analyte. The other component is a polynucleotide that specifically recognizes and binds to a component in a second reagent. In the second step of the procedure, a signalling entity is added. This signalling entity also has two components. The polynucleotide component of the signalling entity binds rapidly and specifically to the complementary polynucleotide component of the first reagent, thereby forming a bridge between the two reagents of the invention. The other component of the second reagent is the signal generating compound of the reagent. Binding of the first reagent to the target, followed by binding of the two complementary polynucleotide portions of the first and second reagents to form the bridge, effectively delivers the signal generating compound to the target. In this reference sequence-specific polynucleotide components are used to rapidly and specifically link two reagents in a multi-step process for detection of target analytes in samples.
Thus, there is a need to develop an in vivo delivery system which exhibits high specificity for target sites, low background levels, and which also minimizes unnecessary exposure of patients to radioactive ions, or other hazardous materials. This type of targeted in vivo delivery system would represent a valuable tool for the diagnosis and treatment of a wide variety of disease states.